The origin of the giant ground Aplothorax burchelli on St Helena Island

TEIJI SOTA1*, MICHIO HORI2, CLARKE SCHOLTZ3, GAYANE KARAGYAN4, HONG-BIN LIANG5, HIROSHI IKEDA6 and YASUOKI TAKAMI7

1Department of Zoology, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606-8502, Japan 2Kyoto University, Sakyo, Kyoto 606-8501, Japan 3Department of Zoology and Entomology, University of Pretoria, Pretoria 0002, Republic of South Africa 4Scientific Center of Zoology and Hydroecology, National Academy of Sciences of the Republic of Armenia, Yerevan 0014, Armenia 5Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China 6Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561, Japan 7Graduate School of Human Development and Environment, Kobe University, Nada, Kobe 657-8501, Japan

*Corresponding author. E-mail: [email protected]

This is the accepted version of the article published in Biological Journal of the Linnean

Society, 2020, 131, 50-60.

1 Abstract

Highly isolated oceanic islands harbor endemic ground that have lost the ability to fly. Here, we investigate the origin of the possibly extinct flightless giant Aplothorax burchelli on St Helena Island in the South Atlantic. A. burchelli was considered to be a member of the subtribe Calosomina (=genus ) of the subfamily (Coleoptera: Carabidae) closely related to the genus Ctenosta

(=Calosoma subgenus Ctenosta), but this proposition was questioned due to its unique external and genital morphology. We conducted a phylogenetic analysis of mitogenome sequences using historical specimens of A. burchelli and samples of representative species of Carabinae. Our analysis of 13 protein coding gene sequences revealed that A. burchelli is definitely a member of Calosomina, most closely related to a species of

Ctenosta. Further analysis using NADH dehydrogenase subunit 5 gene sequences from most groups in Calosomina showed that A. burchelli formed a monophyletic group with

Ctenosta species from Africa and Madagascar. Our results suggest that the ancestor of A. burchelli, which had the ability to fly, colonized St Helena from Africa after the emergence of the island 14 million years ago, and has since undergone evolutionary changes in conjunction with loss of flight.

ADDITIONAL KEYWORDS: Biogeography – Calosoma – colonization – dispersal – flightlessness – oceanic island – mitogenome – molecular phylogeny.

2 INTRODUCTION

Flightless ground beetle species are known to predominate on highly isolated oceanic islands (Darlington, 1943). Such flightless species are likely the descendants of flight-capable species with hind wings, and selection for winglessness has been associated with successful establishment of populations, and subsequent evolution and radiation of endemic species. Precise determination of the origin and evolutionary process of flightless ground beetle species on remote islands requires contemporary molecular phylogenetic analyses, but such studies are limited (e.g., Liebherr &

Maddison, 2013; Maddison, Sproul, & Mendel, 2019).

St Helena is one of the most isolated oceanic islands in the South Atlantic. The nearest continental land is the western African coast, at a distance of ~1,900 km. St

Helena was formed by volcanic activity occurring during the late Miocene; it emerged above the sea surface 14 Ma ago (Ashmole & Ashmole, 2000)(Basilewsky, 1985). The flora and fauna of St Helena include endemic species that often exhibit remarkable characteristics, probably resulting from evolutionary changes since the colonization by ancestral species. The endemic St Helena species are closest to taxonomic groups in

Africa, South America, etc. (Ashmole & Ashmole, 2000), but molecular phylogenetic analyses of the origins of endemic St Helena species have so far been limited.

Unfortunately, extensive habitat destruction and invasion of non-native and plants since the discovery of St Helena in the early 1500s have resulted in the extinction of many native animals and plants (Ashmole & Ashmole, 2000), making their study more difficult.

The giant ground beetle Aplothorax burchelli Waterhouse is a remarkable, large wingless beetle of the subfamily Carabinae (Coleoptera: Carabidae), representative of

3 the unique fauna of St Helena. This species was initially considered to belong to the genus (subtribe ) of the tribe , and Aplothorax was proposed as a subgenus (Waterhouse, 1842). However, subsequent taxonomists considered it a distinct group closer to the genus Calosoma. Jeannel (1940) proposed the Calosomatina

(= Calosomina) which contained 20 genera including Aplothorax (note that the latest revision by Bruschi (2013) treats all groups of Calosomina in the genus Calosoma).

Jeannel (1940) recognized that A. burchelli is close to Ctenosta (= the subgenus

Ctenosta; Bruschi, 2013), based on the characteristics of the male genitalia, and included the genus Aplothorax in Calosomina. However, Basilewsky (1972) disagreed, instead considering Aplothorax a distinct tribe (Aplothoraxini) of the subfamily

Carabinae. Prüser & Mossakowski (1998) conducted a parsimony analysis of morphological characters in the subfamily Carabinae and found that Calosoma

(Calosomina) and Aplothorax were sister groups, but they did not analyze the relationships among Aplothorax and various subgenera of Calosoma. In the review by

Bruschi (2013), which we follow in this paper, all groups in Calosomina were treated as subgenera in the genus Calosoma, but Aplothorax was left out of Calosoma. A molecular analysis of the phylogenetic position of A. burchelli is thus warranted.

Unfortunately, however, live A. burchelli have not been found since 1966–1967, when researchers from the Museum of Central Africa led by P. Basilewsky collected a number of specimens (Basilewsky, 1972). Currently, A. burchelli is considered to be extinct due to the extensive habitat destruction and predation by invasive animals since the discovery of the island in the 16th century (Ashmole & Ashmole, 2000; Gray et al.,

2019).

Historical specimens preserved dry for 50 or more years are frequently used for

4 molecular phylogenetic studies of including carabid beetles. Maddison et al.

(2019) studied the origin of St Helena’s endemic Bembidiini species (Carabidae) by extracting genomic DNA from museum specimens. They showed that the studied species were all placed in the genus Bembidion and formed a monophyletic group with a species from La Rèunion; this clade was sister to the African subgenus Omotaphus, suggesting African origins of St Helena and La Rèunion species.

In this study, we conducted a molecular phylogenetic analysis focusing on mitogenomes of representative species of the subfamily Carabinae (Coleoptera:

Carabidae), including A. burchelli, to reveal its phylogenetic origin. We used specimens collected in 1967 and successfully obtained mitochondrial protein coding gene sequence data. Our results indicate that A. burchelli is a member of the genus Calosoma and closely related to the subgenus Ctenosta, as was suggested by Jeannel (1940).

MATERIAL AND METHODS

DNA EXTRACTION AND SEQUENCING

For the mitogenomic analysis, we used 24 species from all five groups of the subfamily

Carabinae, together with A. burchelli (Table 1). Trachypachus slevini () was used as the outgroup taxon (Table 1). Of the sample specimens, two A. burchelli and one Calosoma chlorostictum were pinned, dry specimens collected on St Helena

Island in 1967 by the Belgian group and given to Professor Emeritus Ryosuke Ishikawa at Tokyo Metropolitan University by P. Basilewsky. These specimens are due to be deposited at the University Museum of the University of Tokyo. Specimens of the other species were preserved in 99% ethanol or RNAlater solution (Invitrogen, Carlsbad, CA,

5 USA). Total genomic DNA was extracted from tissues of ethanol or RNAlater-fixed specimens using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). For pinned specimens, total genomic DNA was extracted from legs without noticeable destruction using the DNeasy Micro Kit (Qiagen) following the manufacturer’s protocol.

The quality and quantity of extracted DNA were assessed using a TapeStation High

Sensitivity D1000 kit (Agilent Technologies, Santa Clara, CA, USA), NanoPhotometer

(Implen, München, Germany), and Qubit (Thermo Fisher Scientific, Waltham, MA,

USA). Shotgun libraries were prepared using the NEBNext Ultra II FS DNA Library

Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) and sequenced on an

Illumina HiSeq X Ten platform (Illumina, San Diego, CA, USA) at Macrogen Co. Ltd.

(Kyoto, Japan).

SEQUENCE DATA ANALYSIS

Raw Illumina reads were filtered with fastp (version 0.20.0; Chen et al., 2018) to remove low-quality and adaptor-containing reads. To assemble mitogenomes directly from the clean reads, we used NOVOplasty (Dierckxsens, Mardulyn, & Smits, 2017) with a seed sequence of the Carabus granulatus mitogenome (GenBank accession no.:

NC044759). If the NOVOplasty assembly failed, we assembled scaffolds from the sequence reads using IDBA-UD (Peng et al., 2012) and searched for mitochondrial

DNA sequences in the scaffolds against the C. granulatus complete mitochondrion sequence (GenBank accession no.: NC044759) using BLASTn (version 2.2.31+;

(Altschul et al., 1997). To obtain mitogenomic sequences for phylogenetic analysis of specimens that failed NOVOplasty assembly, the raw reads were mapped to the reference mitogenome sequence of C. granulatus using Geneious Prime 2020.0 5

6 (Biomatters Ltd., Auckland, New Zealand), and a consensus sequence was obtained for each specimen with IUPAC ambiguity codes or Ns for undetermined or low-coverage sites. All mitogenome sequences thus obtained were aligned to the reference mitogenomes of C. granulatus (GenBank accession no.: NC044759) and a Calosoma sp.

(possibly ; GenBank accession no.: GU176340) using MEGA version X (version 10; (Kumar et al., 2018), and sequences for 13 protein coding genes were extracted for use in subsequent phylogenetic analyses. The cleaned raw reads were deposited at the DDBI Sequence Read Archive (DRA) of the DNA Data Bank of Japan

(DDBJ). Aligned sequence matrices are archived at figshare (doi:

10.6084/m9.figshare.12355844).

PHYLOGENETIC ANALYSIS

We performed a maximum likelihood (ML) analysis with the sequence matrix of 13 protein coding genes using RAxML (version 8.2; Stamatakis, 2014). An optimal partitioning scheme with 24 partitions was obtained from a total of 39 partitions (13 genes × 3 codon positions) using PartitionFinder2 (version 2.1.1; Lanfear et al., 2017), with the GTR+G substitution model used for all partitions. A rapid bootstrap analysis with 1,000 replications was used to obtain the best tree and node support values.

Based on the results of the above analysis, we searched for species closely related to A. burchelli using a collection of NADH dehydrogenase subunit 5 (ND5) gene sequences retrieved from GenBank, together with the ND5 sequences of several

Calosoma species obtained in the present study (see Table S2 for the list of species and sequence accession numbers). Most ND5 gene sequences of Calosoma species were published by Su, Imura, & Osawa (2005). The ND5 dataset contained two sequences of

7 A. burchelli, 46 sequences from 17 of 19 subgenera of Calosoma, and 25 sequences from Carabus (as the outgroup). In total, 73 sequences were used in the phylogenetic analysis of ND5 gene genealogy. With this dataset, a ML analysis was conducted using

RAxML (version 8.2). A rapid bootstrap analysis with 1,000 replications was performed with partitioning of three codon positions, using the GTR+G substitution model for each partition.

DIVERGENCE TIME ESTIMATION

To infer the divergence time of A. burchelli from its closest species, we performed a

Bayesian relaxed clock analysis based on a lognormal clock model, using the

MCMCTree program in the PAML package (version 4.8; Yang, 2007). This analysis used the topology of the best tree obtained from the ML analysis of the 13 protein coding gene sequences. Authentic fossil records of Carabinae are limited to those from the Miocene or later (e.g., Penev, Casale, & Turin, 2003) and not readily used for calibrating internal nodes in divergence time estimation. Therefore, we referred to the clock tree of McKenna et al. (2019) for the split of Trachypachus and Carabinae (170

Ma; 150–197 Ma) and that of Andújar, Serrano, & Gómez-Zurita (2012) for the split of

Carabina and Calosomina (the Carabini stem node) (31 Ma; 24–38 Ma). The age of the

Carabini stem is controversial as it was estimated to be ancient as 104 Ma (the

Cretaceous) when the tree of Carabina taxa was constrained only at the split of

Trachypachus and Carabinae (Toussaint & Gillett, 2017); however, such a calibration strategy leads to unrealistically ancient divergence of Carabus or Calosoma taxa. The estimated divergence time of 24–38 Ma (the Eocene–Oligocene) is more probable for the biogeographic pattern of the Carabini (Andújar, Serrano, & Gómez-Zurita, 2012). In

8 addition to the above two calibration points, we assumed that divergence of

Maoripamborus in New Zealand and Pamborus in Australia occurred >85 Ma ago (Sota et al., 2005). In the divergence time estimation, first, an approximate substitution rate was estimated assuming a putative root age of 170 Ma. Soft bound constraints were used for the three node: (1) divergence between Trachypachidae and Carabinae,

150–197 Ma; (2) divergence between Carabina and Calosomina, 24–38 Ma; and (3) divergence between Pamborus and Maoripamborus, >85 Ma. We performed two independent Markov chain Monte Carlo runs of 50,000 burn-in generations and 500,000 generations, with sampling every 50 generations, and confirmed convergence of the estimated node ages in the two runs.

RESULTS

We obtained 151-bp Illumina paired-end sequence data with 18–106 million reads and total lengths of 2.1–12.6 Gbp for the 25 specimens (Table 1). The NOVOplasty assembly was successful for all but three specimens, including two 52-year-old A. burchelli specimens. We blasted the assembled scaffolds from the two A. burchelli specimens (B89, B90) against the reference mitogenome of C. granulatus (Table S2).

We found that 14 and 10 of B89 and B90 scaffolds with total lengths of 13,141 bp and

8,549 bp, respectively, reliably matched portions of the 16,918-bp C. granulatus mitogenome sequence (Table S2). We then mapped the A. burchelli sequence reads to the C. granulatus mitogenome sequence, and obtained the consensus sequence for each specimen used in the mitogenome alignment with other specimens. In contrast to the A. burchelli specimens, the sequence reads from the St Helena Calosoma chlorostictum

9 specimen (also aged 52 years) could be assembled into a circularized mitogenome sequence. The mitogenome sequences from 25 specimens were aligned with reference sequences of Carabus and Calosoma species, and 13 conserved protein coding gene sequences were extracted for phylogenetic analysis.

The ML tree (Fig. 1) resulting from alignment of the 13 protein coding gene sequences (11,201 bp) clearly showed the relationships among the tribes and subtribes of Carabinae, in which is sister to all other tribes of Carabinae, and Pamborini and Ceroglossini are sister to each other and to Carabini (subtribes Carabini and

Calosomina). Two A. burchelli sequences were located within the subtribe Calosomina

(genus Calosoma) and sister to Calosoma (Ctenosta) senegalense, with 100% bootstrap support.

Among the partial ND5 sequences from 17 of 19 subgenera of the genus

Calosoma (Fig. 2), A. burchelli sequences were grouped with three Ctenosta species (C. senegalense, Calosoma grandidieri, Calosoma planicolle), with 94% bootstrap support.

A. burchelli was closest to C. senegalense among the three Ctenosta species, but the relationships among these species were not supported by the bootstrap analysis. The clade of Ctenosta and Aplothorax was included in a well-supported clade (bootstrap value, 95%) with Campalita, three flightless subgenera (Orinodromus, Carabomorphus, and Carabophanus), Caminara, and a North American Camedula species Calosoma

(Camedula) marginale. Calosoma (Campalita) chlorostictum from St Helena was close to Calosoma (Campalita) maderae and Calosoma (Campalita) auropuncutatum (= C.

(C.) maderae; Bruschi, 2013).

In our divergence time estimation using the three calibration points (Fig. 3), the divergence time of A. burchelli and C. senegalense was 14 Ma (95% CI, 7–21 Ma).

10

DISCUSSION

PHYLOGENETIC POSITION

Our results indicated that A. burchelli is a member of the genus Calosoma (tribe

Carabini: subtribe Calosomina) related to the subgenus Ctenosta (Figs. 1, 2). This finding corroborates Jeannel's (1940) view of the phylogenetic position of Aplothorax, although Jeannel (1940) treated Ctenosta and Aplothorax as genera of Calosomina. In the analysis of ND5 gene sequences, the relationships among A. burchelli and four

Ctenosta species were not resolved, though they formed a monophyletic group with high bootstrap support (Fig. 3). Thus, the exact relationships between A. burchelli and

Ctenosta species should be explored in a future analysis including all Ctenosta species and additional genomic sequence data. Ctenosta comprises nine species (Bruschi, 2013), of which four occur in Africa (Calosoma roeschkei, Calosoma guineense, Calosoma strandi, and Calosoma scabrosum), two in Africa and Madagascar (C. senegalense and

C. planicolle), two in Madagascar only (C. grandidieri and Calosoma bastardi), and one in South Asia (Calosoma orientale). Of these, C. senegalense has the widest distribution range and is a candidate for the closest extant species, as in our study. Other

African species such as C. scabrosum and C. roeschkei, which were not included in our analysis, are also candidates because they show elytral patterns similar to those of A. burchelli.

Another Calosoma species, C. (Campalita) chlorostictum, inhabits St Helena; this species retains its hind wings and can fly. Its distribution range encompasses Africa,

Madagascar, the Middle East, and the isolated South Atlantic islands of St Helena,

11 Ascension, and Tristan da Cunha (Bruschi, 2013). Although we have no ND5 sequence data for African C. chlorostictum specimens, the specimen from St Helena was closely related to C. (Campalita) maderae from Spain and Saudi Arabia. C. maderae has a wide distribution range including West Asia, Europe, North Africa, the Middle East, and the

Canary Islands. The genetic distance between C. maderae and C. chlorostictum (0.0159, uncorrected p) is much smaller than that between Calosoma chinense from East Asia and C. maderae (0.045–0.046), and that between C. senegalense and A. burchelli

(0.062–0.065). These facts suggest a recent divergence of C. maderae and C. chlorostictum, and a relatively recent colonization of C. chlorostictum on St Helena

(Basilewsky, 1972).

CHARACTER EVOLUTION

Our results suggest that the ancestral A. burchelli was a Ctenosta-like Calosoma that was able to fly, with marked morphological evolution subsequently occurring on the isolated island. Calosoma species with hind wings, including Ctenosta species, typically exhibit elytral parts with broad shoulders (Fig. 4). In Calosoma, degeneration of the hind wings and wing muscles (i.e., the evolution of flightlessness) has occurred in a few lineages in different continental areas (Africa, Eurasia, and the Americas), but on an island this phenomenon is limited to A. burchelli. The degeneration of flight muscles and hind wings is associated with a slender body shape with narrow shoulders, which allows allocation of more resources to reproduction. Thus, flightlessness may have been favored in highly isolated habitats where dispersal by flight confers little fitness advantage within a small oceanic island and dispersal out from the island would have no future return. Many endemic beetle species on St Helena Island are similarly flightless

12 (Darlington, 1943; Basilewsky, 1985).

The most conspicuous male genital character of Calosoma species, compared to its sister group Carabus, is the presence of a well-developed ligula in the endophallus and a much less developed aedeagus, which is associated with a less developed endophallus housed in the aedeagus (Ishikawa, 1978). Although the modification of the aedeagus is remarkable in A. burchelli, making this species unlike Calosoma, its endophallus possesses a sclerotized ligula with a curved hook point, similar to that of

Ctenosta species (Jeannel, 1940; Fig. 4). In the subfamily Carabinae, development of the endophallus and the associated enlargement of the aedeagus is seen only in Carabus and some Pamborus (Sota et al., 2005; Takami & Sota, 2006). Other groups, including

Calosoma, have a slender aedeagus with an undeveloped endophallus. The function of the modified aedeagus in A. burchelli and its evolutionary factors are interesting subject for future study.

COLONIZATION OF ST HELENA BY CARABID BEETLES

St Helena Island is a volcanic island that emerged above the sea ~14 Ma ago, and habitable conditions for terrestrial animals may have first occurred 11 Ma ago

(Basilewsky, 1985). In our divergence time estimation, the estimated divergence time of

A. burchelli and C. senegalense was 14 Ma, coinciding with the age of St Helena Island, with the confidence interval of 7–21 Ma. These results may imply that A. burchelli diverged from its sister species (not necessarily C. senegalense) between 7–21 Ma and then colonized on St Helena Island between 7–14 Ma. However, these results were strongly affected by the node-age constraint for the Carabini stem node (see Materials and Methods) and should be interpreted cautiously. We need well-evidenced calibration

13 points for estimation of divergence times in the Calosomina.

We infer that the ancestor of A. burchelli could fly as do Ctenosta species, and that the ancestral A. burchelli may have achieved colonization by flight and wind-borne dispersal. In fact, some Calosoma beetles appear to have achieved long distance dispersal by flight, such as from North Africa to Europe (Bruschi 2013). Other ways of colonization, such as rafting on a large log, etc., may not be ruled out completely, but this possibility would be small given the long journey in the sea water until reaching the oceanic island. On the other hand, C. (Campalita) chlorostictum appears to be a much more recent colonizer of St Helena, given the minimal genetic divergence from its related species. For this species, even the possibility of accidental introduction by ships after the discovery of the island (in 1502) cannot be ruled out.

The endemic carabid fauna of St Helena includes flightless species of the tribe

Bembidiini, which are called “St Helena Peaks Bembidion” and the sister group of the subgenus Omotaphs in Africa (Maddison et al. 2020). Bembidion beetles are generally small and active flyers, and long distance (probably wind-borne) dispersal from the southwest Pacific to the Society and Hawaiian Islands has been proposed (Liebherr &

Maddison, 2013). The ancestral Bembidion species might also have colonized St Helena from Africa by wind-borne dispersal. It is interesting to estimate the divergence time of

St Helena Peaks Bembidion species from their sister group for understanding the colonization history of carabids on St Helena Island.

CONCLUSION

Using mitogenome data, our study resolved the origin of A. burchelli and confirmed that

14 it is a member of the genus Calosoma, closely related to the subgenus Ctenosta. The ancestral species was probably winged, lost its flight muscles and hind wings, and evolved a modified aedeagus during adaptation to the island habitat. Thus, A. burchelli represents a good example of the evolution of flightless beetles in an isolated island habitat. Genomic comparison of A. burchelli and its sister species in the subgenus

Ctenosta may reveal the genetic changes that contributed to the evolution of flightlessness and subsequent morphological evolution, including of the genitalia. Our results demonstrate the utility of DNA extracted from historical specimens of A. burchelli, and this line of genomic study could be extended to nuclear genomic DNA.

On the other hand, it may be too early to conclude that this beetle is extinct. A. burchelli is nocturnal and hides underground during daytime; the beetles are active only after substantial rainfall and may be completely inactive during the dry period of the year

(Basilewsky, 1985). The chance of encountering or collecting this species is thus limited; indeed, since the time of Napoleon’s exile in the early 1800s, very few specimens had been collected before the expedition of the Belgian team in 1966–1967; thus, there is a faint possibility that it has survived.

ACKNOWLEDGMENTS We thank Professor Emeritus R. Ishikawa for continual support and allowing the use of specimens for DNA extraction; D. Maddison, L. Bocak, Y. Okuzaki, A. Casale, J.

Galian, P. Oromí, J. Yoshimura, S. Kakishima, D. Pearson, M. Genich, K. Fukumori, K.

Miyashita, and T. Hikida for helping with sampling; New South Wales National Parks and Wildlife Service, Queensland Parks and Wildlife Service, Department of

15 Conservation, New Zealand Government, La Corporación Nacional Forestal, Chile,

Cabildo de Tenerife, Ministry of Environment and Tourism, Government of Namibia, and Lake Tanganyika Research Unit, Department of Fisheries, Zambia for research permission. T.S. thank L. Malan, R. Cairns-Wicks, A. Dutton, L. Fowler and R. Key for discussion about the giant ground beetle research, D. Pryce and the St Helena National

Trust for the photograph, and T. Nakano for taxonomic discussion. This study was supported by JSPS KAKENHI (nos. 11304056, 17405007, 23405009, 15H02637).

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Fig. 1. Phylogenetic relationships among the groups (tribes) of the subfamily Carabinae

and the position of Aplothorax burchelli. Photo credit: T. Sota.

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Fig. 2. Phylogenetic tree based on mitochondrial ND5 gene sequences of Calosoma

(subtribe Calosomina) species and species from the sister group Carabus (subtribe

Carabina). The hind wing condition follows the description in Bruschi (2013).

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Fig. 3. Divergence time of the Carabinae. Nodes with closed triangles are constrained

(see Material and Methods for details). Bars indicate 95% confidence intervals.

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Fig. 4. Habitus and male genitalia. (a) Aplothorax burchelli, male; (b) A. burchelli,

female; (c) Calosoma (Ctenosta) senegalense, male; (d) Calosoma (Ctenosta)

planicolle, male; (e) Calosoma (Ctenosta) grandidieri, male; (f) apex of the

aedeagus of A. burchelli male; enlargement of the abdominal tip of (a); (g) male

genitalia of A. burchelli (redrawn from Jeannel, 1940; p. 134, fig. 96–98), (h) male

genitalia of C. (C.) senegalense. Photo credit: (a) and (f), D. Pryce/the St Helena

National Trust; (b)–(e), (g) and (h), T. Sota.

23 Table 1. List of specimens used in the mitogenome study.

Sequence reads1 Accession Mitogenome Species Sample Locality Year of No. reads, Total Mean BioSample ID2 Assembled bp3 ID collection ×106 length, Gb length, bp

Aplothorax burchelli B89 St Helena I. 1967 22.9470 2.3203 101 SAMD00208720 (15825)

Aplothorax burchelli B90 St Helena I. 1967 48.1959 5.6238 116 SAMD00208721 (15834)

Tribe Carabini Subtribe Calosomina Calosoma breviusculum B55 Shirak, Armenia 2013 50.7072 6.2075 122 SAMD00208715 16457 Calosoma chlorostictum B96 St Helena I. 1967 72.3468 6.9062 95 SAMD00208722 17110 Calosoma imbricatum B45 Northern Cape, South Africa 2017 27.5755 3.5488 128 SAMD00208708 (16474) B43 Hokkaido, Japan 2016 27.9983 3.5649 127 SAMD00208707 16426 Calosoma marginale B52 Texas, USA 2012 18.4811 2.1390 115 SAMD00208713 16433 B51 Grenada, Mississippi, USA 2015 28.0091 3.2591 116 SAMD00208712 16482 Calosoma senegalense B50 Hardap, Namibia 2017 46.7255 5.8125 124 SAMD00208711 16458 Calosoma subaeneum B54 Washington, USA 2016 105.7605 12.5722 118 SAMD00208714 16456 Tribe Carabini Subtribe Carabina Carabus davidis A01 Jiangxi, China 2006 36.2739 4.8191 132 SAMD00208696 16625 Carabus maeander A17 Hokkaido, Japan 1998 35.6161 4.3731 122 SAMD00208698 16646 Carabus abbreviatus A21 Tenerife I., Spain 2017 41.6532 5.4019 129 SAMD00208700 16600 Carabus genei A18 Sardinia I., Italy 2017 47.2469 6.2459 132 SAMD00208699 16547

24 Carabus intricatus A05 Moravia, Czech 2015 47.5110 6.1245 128 SAMD00208697 16574 Carabus melancholicus A32 Madrid, Spain 1996 59.0931 7.5855 128 SAMD00208701 16802 Tribe Ceroglossini

Ceroglossus darwini B38 Puyehue, Chile 2015 44.0739 6.0803 137 SAMD00208705 16467 suturalis B40 Mallaganes, Chile 2015 44.5999 5.5543 124 SAMD00208706 16474 Tribe Cychrini

Cychrus caraboides B31 Val Sessera, Italy 2000 45.3358 5.8602 129 SAMD00208702 17343 petersi B35 Arizona, USA 2016 51.2401 7.2032 140 SAMD00208703 16844 Sphaerodrus canadensis B36 Virginia, USA 2016 42.1741 5.6247 133 SAMD00208704 16885 Tribe Pamborini

Maoripamborus fairburni B73 Waipaua, New Zealand 2002 44.1781 6.2910 142 SAMD00208719 17025 Pamborus alternans B57 New South Wales, Australia 2001 60.3485 6.9825 115 SAMD00208717 16668 Pamborus punctatus B67 Mt.Hypipamee, Australia 2001 34.9593 4.5790 130 SAMD00208718 16798 Family Trachypachidae

Trachypachus slevini B56 Oregon, USA 2018 42.2076 5.2948 125 SAMD00208716 17173

1, Sequence reads after filtering. 2, DDBI/DRA BioSample IDs under BioProject PRJDB9367. 3, Length of the whole mitogenome resulting from NOVOplasty assembly. Sequence lengths of B89, B90 and B45 obtained by mapping on the reference genome are in parentheses.

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